Hypersensitivity of myelinated A-fibers via toll-like receptor 5 promotes mechanical allodynia in tenascin-X-deficient mice associated with Ehlers–Danlos syndrome

Deficiency of an extracellular matrix glycoprotein tenascin-X (TNX) leads to a human heritable disorder Ehlers–Danlos syndrome, and TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. We previously reported that TNX-deficient (Tnxb−/−) mice exhibit mechanical allodynia and hypersensitivity to myelinated A-fibers. Here, we investigated the pain response of Tnxb−/− mice using pharmacological silencing of A-fibers with co-injection of N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314), a membrane-impermeable lidocaine analog, plus flagellin, a toll-like receptor 5 (TLR5) ligand. Intraplantar co-injection of QX-314 and flagellin significantly increased the paw withdrawal threshold to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not 5 Hz (C fiber responses) in wild-type mice. The QX-314 plus flagellin-induced silencing of Aδ- and Aβ-fibers was also observed in Tnxb−/− mice. Co-injection of QX-314 and flagellin significantly inhibited the mechanical allodynia and neuronal activation of the spinal dorsal horn in Tnxb−/− mice. Interestingly, QX-314 alone inhibited the mechanical allodynia in Tnxb−/− mice, and it increased the paw withdrawal threshold to stimuli at frequencies of 250 Hz and 2000 Hz in Tnxb−/− mice, but not in wild-type mice. The inhibition of mechanical allodynia induced by QX-314 alone was blocked by intraplantar injection of a TLR5 antagonist TH1020 in Tnxb−/− mice. These results suggest that mechanical allodynia due to TNX deficiency is caused by the hypersensitivity of Aδ- and Aβ-fibers, and it is induced by constitutive activation of TLR5.

Tenascin-X (TNX) is an extracellular matrix glycoprotein, and TNX deficiency caused by compound heterozygous and homozygous mutations of the gene leads to a heritable connective tissues disorder Ehlers-Danlos syndrome (EDS) [1][2][3][4][5] .EDS is characterized by hyperextensibility of skin, hypermobility of joints, and fragility of various connective tissues.In addition, pain is a sever clinical manifestation of EDS patients, and approximately 90% of EDS patients complain of chronic pain [6][7][8] .EDS patients suffer from chronic neuropathic pain, generalized body pain, gastrointestinal pain, headache, dysmenorrhea, and fatigue, in addition to connective tissues-related pain such as joint pain, soft-tissue pain, and dislocations 6,7 .TNX-deficient EDS patients complain of chronic pain such as chronic joint pain, myalgia, abdominal pain, fatigue, paresthesia, and axonal polyneuropathy 3,5 .We have previously reported the pain behaviors of TNX-deficient (Tnxb −/− ) mice as a murine EDS model 9 .Tnxb −/− mice exhibited increased sensitivity to innocuous mechanical stimuli, which is called mechanical allodynia, a major feature of neuropathic pain.Tnxb −/− mice also showed significant hypersensitivity to transcutaneous sine wave stimuli at frequencies of 250 Hz (myelinated Aδ fiber responses) and 2000 Hz (myelinated Aβ fiber responses) using analysis of sensory afferent fiber responses to a sine wave electric stimulator.However, the hypersensitivity of myelinated Aδ-and Aβ-fibers by which TNX deficiency complicates the mechanical allodynia remains unknown.
It has been reported that blocking A-fibers can be specifically achieved by using the toll-like receptor 5 (TLR5) ligand flagellin and the membrane-impermeable sodium channel blocker N- (2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) 10 .Activation of TLR5 induced by flagellin results in neuronal entry of QX-314, provoking TLR5-dependent blockade of sodium currents, primarily in Aβ fibers.Intraplantar co-administration of flagellin and QX-314 suppressed mechanical allodynia in neuropathic pain models induced by chemotherapy, nerve injury, and diabetic neuropathy.On the other hand, blocking C-fibers can be specifically achieved using the transient receptor potential vanilloid 1 (TRPV1) ligand capsaicin and QX-314 11 .Co-administration of capsaicin and QX-314 resulted in the neuronal entry of QX-314 through the ion channel TRPV1, provoking a blockade of C-fibers.
In the present study, we used fiber-specific blockade using co-injection of QX-314 with flagellin or capsaicin to Tnxb −/− mice, in order to elucidate the correlation between TNX deficiency-induced hypersensitivity of myelinated A-fibers and the mechanical allodynia.We examined the combination of sensory afferent fiber responses to a sine wave electric stimulator and behavioral pharmacology in Tnxb −/− mice injected with QX-314, flagellin, or capsaicin.We demonstrated that TNX-deficiency-induced mechanical allodynia is mediated by A-fiber hypersensitivity, and that QX-314 alone-inhibited mechanical allodynia is mediated by constitutive activation of TLR5 in Tnxb −/− mice.

Discussion
In the present study, we revealed that the mechanical allodynia and central sensitization of the spinal dorsal horn were medicated by the hypersensitization of Aβ and Aδ fibers in Tnxb −/− mice, by using pharmacological sensory fiber silencing.A-fiber silencing, intraplantar (i.pl.) co-administration of QX-314 and flagellin, significantly increased the paw withdrawal threshold of Aβ and Aδ fibers to transcutaneous sine wave stimuli at frequencies of 250 Hz and 2000 Hz, respectively, in wild-type mice.Intraplantar co-administration of QX-314 and flagellin significantly increased the paw withdrawal threshold of Aβ and Aδ fibers in Tnxb −/− mice, and it inhibited the mechanical allodynia and spinal neuronal activation in Tnxb −/− mice.Notably, in Tnxb −/− mice, but not wild-type mice, i.pl.administration of QX-314 alone significantly increased the paw withdrawal threshold of Aβ and Aδ fibers and subsequent mechanical allodynia.Thus, the activation of Aβ and Aδ fibers led to the TNX deficiency induced-mechanical allodynia.Furthermore, the QX-314-induced inhibition of mechanical allodynia was reduced by TLR5 antagonist TH1020, suggesting that it is mediated via constitutive activation of TLR5.Blocking A-fibers can be specifically achieved by using TLR5 ligand flagellin and the membrane-impermeable sodium channel blocker QX-314 10 .Likewise, i.pl.co-administration of QX-314 and flagellin significantly increased the paw withdrawal threshold of Aβ and Aδ fibers to transcutaneous sine wave stimuli at frequencies of 250 Hz and 2000 Hz in wild-type mice (Fig. 1).The QX-314 plus flagellin-induced increase of threshold to stimuli at frequencies of 250 Hz and 2000 Hz was also observed in Tnxb −/− mice (Fig. 4).We have previously reported that TNX deficiency induces mechanical allodynia and central sensitization in the dorsal horn of the spinal cord, in addition to hypersensitization of myelinated Aβ and Aδ fibers 9 .A-fiber silencing, i.pl.co-administration of QX-314 and flagellin, significantly inhibited the mechanical allodynia (Fig. 2) and the increase of c-Fos positive neurons of spinal dorsal horn (Fig. 3).Surprisingly, intraplantar administration of QX-314 alone significantly increased the paw withdrawal threshold to stimuli at frequencies of 250 Hz and 2000 Hz in Tnxb −/− mice (Fig. 4) but not wild-type mice (Fig. 1).Intraplantar administration of QX-314 alone inhibited the mechanical allodynia in Tnxb −/− mice (Fig. 2).These results suggest that hypersensitization of myelinated Aβ and Aδ fibers responded to stimuli at frequencies of 250 Hz and 2000 Hz led to the mechanical allodynia.
QX-314 is a membrane-impermeable voltage-gated sodium channels blocker that blocks these channels by binding to their intracellular domains.Although the extracellular application of QX-314 theoretically has no effect on the activity of sodium channels, QX-314 has been shown to enter the cytoplasm of cells by activating the TRPV1 channel 11 .Intraplantar co-administration of QX-314 (2% (approximately 60 mM), 10 μL) and capsaicin (1 μg/μL, 10 μL) increased the mechanical threshold for paw withdrawal using von Frey filaments, whereas i.pl.administration of QX-314 alone had no significant effect on the mechanical threshold.In this study, i.pl.administration of QX-314 (30 mM) without a TRPV1 agonist inhibited mechanical allodynia in Tnxb −/− mice (Fig. 2b).Consistent with this result, QX-314 (2%, 60 mM) alone applied i.pl. to UV-burn-induced inflamed paws has an inhibitory effect on paw mechanical sensitivity 19 .During tissue damage and inflammation, inflammatory mediators such as ATP, prostaglandins, and bradykinin released from surrounding damaged or inflamed tissues and the acidic environment surrounding the inflamed tissues have been reported to sensitize the response of TRPV1 20,21 .Furthermore, systemic intraperitoneal administration of QX-314 (1-3 mg/kg) alone reduced bone cancer pain-related behaviors by inhibiting TRPV1-expressing afferents 22 .In a bone cancer pain model, tumor growth induces inflammation and an acidic environment is induced by activated osteoclasts 23 .These reports suggest that the inhibitory effects of QX-314 alone are mediated by TRPV1 activation.However, the TRPV1 antagonist capsazepine failed to suppress the inhibition of mechanical allodynia induced by i.pl.administration of QX-314 alone in Tnxb −/− mice (Figs.5d,e), suggesting that the inhibitory mechanisms of QX-314 alone are independent of TRPV1 channel activation.We identified a novel mechanism of action for i.pl.QX-314 alone, in which TLR5 was involved in inhibiting mechanical allodynia in Tnxb −/− mice.A potent and selective TLR5/flagellin complex antagonist, TH1020, blocked the QX-314-induced inhibition of mechanical allodynia in Tnxb −/− mice (Figs.5a,b).Flagellin, a TLR5 agonist, induces the entry of extracellular QX-314 into the cytoplasm via unidentified pores such as channel 10 .Thus, TNX-deficiency may lead to constitutive activation of TLR5 and subsequent cellular signaling.
Axonal polyneuropathy occurs in TNX-deficient EDS patients 3,5,24 .Nerve conduction studies were abnormal in 80% of TNX-deficient EDS patients, and fulfilled the criteria of sensorimotor axonal polyneuropathy in 40% of the patients 5 .Electromyography shows TNX-deficient EDS patients had a predominant neurogenic and a mixed myogenic-neurogenic patterns 5 .Neuropathic symptoms including paresthesia are frequently observed in patients with TNX-deficient EDS 3,5 .Voermans et al. reported histological changes in sciatic nerves such as mildly smaller inner and outer diameters of the myelinated fiber and reduced collagen fibril density of the endoneurium in Tnxb −/− mice 25 .We have reported that TNX deficiency induces hypersensitivity of myelinated Aβ and Aδ fibers, whereas it has no effect on C-fiber response under the basal condition 9 .In this study, A-fiber silencing, co-administration of QX-314 and flagellin, affected the paw withdrawal threshold of C fiber to stimuli at frequencies of 5 Hz in Tnxb −/− mice (Fig. 4a).This finding raises a possibility that A fibers partially cross over C fiber in sensory fibers of Tnxb −/− mice.Fiber interaction such as ephaptic and cross-excitation is thought to be one of peripheral neuropathy mechanisms whereby nociceptive fibers such as C-and Aδ-fibers could be stimulated by activity in low-threshold mechanoreceptors in non-nociceptive Aβ fibers [26][27][28][29] .TNX-deficiency may induce physical crosstalk between A fibers and C fibers.In addition, C-fiber silencing effect of the paw withdrawal threshold for responses to 5 Hz stimuli in Tnxb −/− mice was lower than that in wild-type mice, although administration of QX-314 plus capsaicin significantly increased the threshold at a frequency of 5 Hz in both wild-type and Tnxb −/− mice (Figs.1a and 4a).There was no difference in the threshold for responses to 5 Hz stimuli at pre-administration between wild-type and Tnxb −/− mice.These results suggest that TNX deficiency affects the C-fiber silencing mechanisms by using QX-314 and capsaicin.The expression level of the capsaicin receptor TRPV1 mRNA in dorsal root ganglion of Tnxb −/− mice was similar to that of wild-type mice (Fig. 5f).TNX-deficient mice showed normal behavioral response to the noxious thermal stimulation in hot plate-test 9 , whereas TRPV1-deficient mice showed impaired behavioral response to the noxious thermal stimulation compared to wild-type mice 30 .Taken together, TNX deficiency inhibits the QX-314 plus capsaicin-evoked C-fiber silencing through an undetermined mechanism independent of TRPV1.The pathophysiological mechanism of peripheral neuropathy in patients with TNX-deficient EDS seems to be related to the dysfunction of unmyelinated C-fibers, in addition to the hypersensitivity of myelinated Aβ and Aδ fibers.
In conclusions, we demonstrated that the TNX deficiency-induced mechanical allodynia is mediated by A-fiber hypersensitivity using pharmacological sensory fiber silencing with co-administration of QX-314 and flagellin.In addition, QX-314 alone led to the silencing of A-fiber responses and inhibition of mechanical allodynia through the constitutive activation of TLR5 in Tnxb −/− mice.Our findings will be useful for managing neurological complications in patients with EDS.

Animals
All experiments were approved by the Animal Experimentation Committee of the Osaka Institute of Technology and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ethical guidelines of the Ethics Committee of the International Association for the Study of Pain.This study was conducted in accordance with ARRIVE guidelines.Tnxb −/− mice were generated as previously reported 31 , and they were backcrossed with C57BL/6 J mice for ten generations.Age-matched C57BL/6 J mice were used as wild-type (Tnxb +/+ ) mice.Seven to 9-week-old male mice were used for the experiments, because of the marked sex-related differences in mechanisms of pain hypersensitivity 32 .Animals were housed under conditions of a 12-h light/12-h dark cycle and at a constant temperature of 22 ± 2 °C.Animals were allowed free access to food and water prior to testing.

von Frey test
Mice were randomly placed in glass chambers on a mesh floor.Mice were habituated to the test environment for 30 min.Mechanical nociception was assessed using the up-down method with von Frey filaments 33 .Mechanical sensitivity was evaluated using calibrated von Frey filaments (0.02-2.0 g).The first stimulus was always a 0.4-g filament.When a paw withdrawal reflex of the paw was elicited, the next lower-rated filament was applied, and when there was no response, the next higher-rated filament was used.After the first change in the response direction, four additional measurements were carried out, and the 50% withdrawal threshold was calculated using the up-down method 33 .

Electrical stimulation-induced paw withdrawal test
The electrical stimulation-induced paw withdrawal test was performed as previously described 22 .Briefly, electrodes (3 mm in diameter) were attached to the left plantar and dorsal surfaces of the hind paws of each mouse.Transcutaneous nerve stimuli using each of three sine wave currents (5, 250, and 2000 Hz) were applied for 3 s through the electrodes.The current intensity increased gradually, and the minimum current intensity of the paw withdrawal response was defined as the paw withdrawal threshold.Transcutaneous nerve stimuli using each sine wave currents were applied to the paw at 5-min intervals.

Immunohistochemistry
Immunohistochemistry was performed as previously described 34 .Briefly, mice were deeply anesthetized using sodium pentobarbital and then intracardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).The lumbar spinal cord was dissected, fixed in 4%